In damaged skin, regeneration is not the same as filling and resurfacing. Healing by scarring results from the faulty, exuberant reconstruction of the dermal architecture and the formation of an epidermis lacking appendages. The overriding hypothesis of this multi-investigator proposal is that the MRL/MpJ mouse, in which ear wounds regenerate, expresses proteins during wound healing that favor regeneration. This concept is bolstered by our recently published work and our current findings that the MRL wound proteome has distinct differences from other mouse strains. We have published evidence that the bone marrow is a rich source of wound fibroblasts, and mesenchymal stem cells (MSC) from the MRL mouse enhance healing due in part to elevated sFRP-1. The first goal is to identify the key proteomic differences between regenerating and non-regenerating wounds within the MRL and between strains, using a novel, precise surgical method with the free-electron laser. Analysis will be accomplished with state of the art, high-resolution proteomic techniques and verified by immunohistochemistry. The second goal is to determine how the migration and differentiation of several stem cell populations is affected by known and newly-identified, regeneration-specific factors in a novel, microfluidic device that can generate complex, two-dimensional concentration gradients. Further validation will use novel wound chambers. The third goal is to build a scaffold, based on a novel polyurethane chemistry, to provide a three-dimensional environment into which factors that recruit stem cells or promote stem cell activity can be released, under controlled conditions, to drive a regenerative response in skin that normally heals by scarring. The project will test known candidates and then those newly identified. The key criteria for regeneration will be restoration of connective tissue architecture, including the formation of functional elastic fibers and the initiation of hair follicle formation. The translational outcome of this joint venture will be the identification of regeneration-promoting molecules, the use of bone marrow-derived cells to promote regeneration, the development of a new device for studying cell behavior, and the production of a bioactive scaffold for recruitment, differentiation, and delivery of morphogens that promote fully functional repair. This will be accomplished by the collaboration of a seasoned team of investigators with expertise in tissue analysis, proteomics, stem cell biology, microfluidics, and polymer chemistry. PUBLIC HEALTH RELEVANCE: Wounds and burns frequently scar and fail to regenerate the original architecture of the skin. Certain strains of mice heal skin much better in some regions of their body, and part of this may be due to differences in their circulating stem cells or the way they are recruited to the wound. This is a study to identify the cell signals that differ between normal healing and regeneration, to see how they affect the behavior of stem cells, and to devise a temporary, synthetic scaffold that can deliver these regenerative signals to restore skin structure and function.